Human glycoprotein

ABSTRACT

The present invention provides polynucleotides which identify and encode a novel human glycoprotein (SC2H). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding SC2H. The invention also provides for the use of substantially purified SC2H and its agonists in the commercial production of recombinant proteins for the treatment of diseases associated with the expression of SC2H. Additionally, the invention provides for the use of antisense molecules to SC2H in the treatment of diseases associated with the expression of SC2H. The invention also describes diagnostic assays which utilize diagnostic compositions comprising the polynucleotides which hybridize with naturally occurring sequences encoding SC2H and antibodies which specifically bind to the protein.

This application is a divisional application of U.S. application Ser.No. 08/695,355, filed Aug. 9, 1996, now U.S. Pat. No. 5,821,086.

FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequencesof a novel human glycoprotein and to the use of these sequences in thediagnosis, study, prevention and treatment of disease.

BACKGROUND OF THE INVENTION

SC2 is a 308 residue glycoprotein highly expressed in neuronal-enrichedregions of the rat central nervous system (CNS). The SC2 sequence has anunglycosylated molecular mass of 36.1 kdal and contains a putativemembrane-spanning domain located near the carboxy terminus (Johnston IGet al (1992) J Neurosci Res 32:159-166). All three potential N-linkedglycosylation sites are on the amino-terminal side of themembrane-spanning domain. Two tyrosine residues are present on thecarboxy-terminal side of the transmembrane domain. SC2 mRNA is presentthroughout postnatal cerebellar development. The highest levels of SC2expression are found in neuronal cell types, including pyramidal cellsof the hippocampus, all layers of the neocortex, granule, and Purkinjeneurons of the cerebellum. SC2 is also expressed at low levels in manyother tissues.

Glycoproteins participate in the mechanisms of neurotransmitter release.The tightly-regulated synaptic vesicle cycle at the nerve terminalconsists of the formation of synaptic vesicles, the docking of vesiclesto the presynaptic plasma membrane, the fusion of these membranes andconsequent neurotransmitter release, endocytosis of the empty vesiclesand the regeneration of fresh vesicles. A variety of glycoproteins areinvolved in these processes, serving as recognition and/or adhesionmolecules (reviewed in Sudhof TC (1995) Nature 375:645-653; Bennet MKand RH Scheller (1994) Curr Opin Neurobiol 4:324-329).

Glycoproteins present in the synapses of the mammalian CNS play a majorrole in the establishment and in the adhesive stability of synapticcontacts and in neuronal functioning. Synaptic modifications induced bylearning and long-term potentiation (LTP) may involve the action of celladhesion molecules, which include a variety of neuronal glycoproteins(Nosten-Bertrand M et al (1996) Nature 379:826-829). Cell adhesionglycoprotein molecules are involved in target recognition andsynaptogenesis at neuromuscular junctions, and may play a role infine-tuning nerve terminal arborization and in modifying the developmentof presynaptic functions. Defects in fasciclin I (Fas I), an insectglycoprotein expressed in motor nerve axons and terminals, result indefective presynaptic function (Zhong Y and Shanley J (1995) J Neurosci15:6679-6687).

The processes of learning and memory establishment involve theglycoprotein-mediated structural remodeling and stabilization ofsynapses. Training chicks in a one-trial passive avoidance task resultsin a cellular cascade which includes 1) phosphorylation of thepresynaptic protein kinase C substrate B-50, 2) increased synthesis ofpre- and postsynaptic glycoproteins, 3) subsequent increases indendritic spine densities, synapse and synaptic vesicle numbers, and 4)prolonged increase in neuronal bursting (Rose SP (1991) Trends Neurosci14:390-397).

Glycoprotein-associated effects are regulated by post-translationalmodifications, such as variations in the structure of attachedcarbohydrate moieties and in phosphorylation. For example, certainsynaptic glycoproteins show enhanced fucosylation in day-old chickssubjected to a passive-avoidance task. Antibodies directed against theseglycoproteins cause a loss of memory of this learned response (MileusnicR et al (1995) J Neurochem 64:2598-2606). Tyrosine phosphorylation ofneuronal glycoproteins paralleling synaptic formation suggests a rolefor these glycoproteins in synapse development and in signaltransduction (Soulliere J et al (1994) J Neurosci Res 37:506-514).

Neuronal atrophy and synapse loss has been correlated with numerousneurodegenerative disorders. The severity of Parkinson diseasecorrelates with the degree of neuronal loss in the substantia nigra. Theprincipal pathologic feature of Huntington disease is severedegeneration of the basal ganglia, which contain a preponderance ofGABA-nergic neurons. Lower and upper motor neuron degeneration is theprincipal pathologic feature of amyotrophic lateral sclerosis (ALS, LouGehrig disease) (Boss B J et al (1994) in Pathophysiology, McCance K Land Huether S E eds, Mosby-Year, St. Louis Mo., pp.527-586).Dementia-associated disorders also involve nerve cell atrophy anddegeneration. Synapse loss in brain tissue correlates with the severityof dementia in Alzheimer's disease (Lassmann H et al (1993) Ann NY AcadSci 695:59-64). viral pathogens attach to host cell surfaces by theinteraction of viral envelope glycoproteins with host membraneglycoproteins. This viral attachment is the first step in the infectivecycle for numerous pathogens, including those which attack the nervoussystem such as poliovirus, rabies, herpes simplex, and HIV. Host cellsurface glycoproteins thus provide targets for the design of anti-viraltherapeutics.

Glycoproteins participate in formation and maintenance of neurons andsynapses and in synaptic vesicle cycling. They are implicated in theacquisition of memory and learning. Understanding the structure andfunction of neuronal glycoproteins will provide insight into normalneuronal and synaptic function and their physiological and pathologicalmodifications. The selective modulation of glycoprotein expression mayprovide a means for the regulation and maintenance of neurons, synapsesand synaptic vesicles in neurodegenerative disorders, as well as in thecontrol of host cell invasion by viral pathogens.

SUMMARY OF THE INVENTION

The present invention discloses a novel human glycoprotein, hereinafterreferred to as SC2H, having homology to the SC2 synaptic glycoproteinfrom rat. Accordingly, the invention features a substantially purifiedglycoprotein, encoded by amino acid sequence of SEQ ID NO:1, havinghomology to rat glycoprotein SC2.

One aspect of the invention features isolated and substantially purifiedpolynucleotides which encode SC2H. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2. In addition,the invention features nucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention further relates to nucleic acid sequence encoding SC2H,oligonucleotides, peptide nucleic acids (PNA), fragments, portions orantisense molecules thereof. The present invention also relates to anexpression vector which includes polynucleotide encoding SC2H and itsuse to transform host cells or organisms. The invention also relates toantibodies which bind specifically to the glycoprotein of SEQ ID NO:1and to a pharmaceutical composition comprising a substantially purifiedglycoprotein of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) and thenucleic acid sequence (SEQ ID NO:2) of the human glycoprotein SC2H,produced using MACDNASIS software (Hitachi Software Engineering Co Ltd,S. San Francisco Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among SC2H (SEQID NO:1), rat SC2(GI 256993; SEQ ID NO:3), and yeast SC2 (GI 1077162;SEQ ID NO:4) produced using the multisequence alignment program ofLASERGENE software (DNAStar Inc, Madison Wis.).

FIG. 3 shows the hydrophobicity plot (generated using MACDNASISsoftware) for SC2H, SEQ ID NO:1; the X axis reflects amino acidposition, and the negative Y axis, hydrophobicity.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,amino acid sequence as used herein refers to peptide or proteinsequence.

"Peptide nucleic acid" as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen P E et al(1993) Anticancer Drug Des 8:53-63).

A "variant" of SC2H is defined as an amino acid sequence that isdifferent by one or more amino acid "substitutions". The variant mayhave "conservative" changes, wherein a substituted amino acid hassimilar structural or chemical properties, eg, replacement of leucinewith isoleucine. More rarely, a variant may have "nonconservative"changes, eg, replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, LASERGENE software.

The term "biologically active" refers to an SC2H having structural,regulatory or biochemical functions of the naturally occurring SC2H.Likewise, "immunologically active" defines the capability of thenatural, recombinant or synthetic SC2H, or any oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells and tobind with specific antibodies.

The term "derivative" as used herein refers to the chemical modificationof a nucleic acid encoding SC2H or the encoded SC2H. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group. A nucleic acid derivative would encode a polypeptidewhich retains essential biological characteristics of natural SC2H.

As used herein, the term "substantially purified" refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

"Stringency" typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe) to about 20° C. to 25° C. below Tm. As willbe understood by those of skill in the art, a stringency hybridizationcan be used to identify or detect identical polynucleotide sequences orto identify or detect similar or related polynucleotide sequences.

The term "hybridization" as used herein shall include "any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing" (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.). Amplification as carried out in the polymerasechain reaction technologies is described in Dieffenbach CW and GSDveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.).

A "deletion" is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring SC2H.

A "substitution" results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

Description

The present invention relates to a novel human glycoprotein, SC2H,initially identified among the partial cDNAs from a human brain tissuelibrary (BRAINOT03) and to the use of the nucleic acid and amino acidsequences disclosed herein in the study, diagnosis, prevention andtreatment of disease. Northern analysis using the LIFESEQ™ database(Incyte Pharmaceuticals, Palo Alto Calif.) indicates that SC2H-encodingnucleotide sequences are abundantly transcribed in brain and othernervous system tissues, both normal and cancerous. SC2H transcripts arehighly abundant in a cDNA library prepared from brain tissue of anAlzheimer's disease patient. SC2H is also transcribed in a variety ofnormal and cancerous tissues including prostate, lung, breast, testis,ovary, thyroid and lymphocytes.

The present invention also encompasses SC2H variants. A preferred SC2Hvariant is one having at least 80% amino acid sequence similarity to theSC2H amino acid sequence (SEQ ID NO:1), a more preferred SC2H variant isone having at least 90% amino acid sequence similarity to SEQ ID NO:1and a most preferred SC2H variant is one having at least 95% amino acidsequence similarity to SEQ ID NO:1.

The nucleic acid sequence encoding a portion of SC2H was firstidentified in the cDNA, Incyte Clone 659029, through acomputer-generated search for amino acid sequence alignments. Thenucleic acid sequence, SEQ ID NO:2, disclosed herein (FIGS. 1A, 1B, and1C) encodes the amino acid sequence, SEQ ID NO:1, designated SC2H. Thepresent invention is based in part on the structural homology shown inFIGS. 2A and 2B among SC2H and other glycoproteins including rat SC2 (GI256993; Johnston, supra) and yeast SC2 (GI 1077162; Andre B et al (995)unpublished).

SC2H consists of 308 amino acids and is a member of the class oftransmembrane glycoproteins including SC2. From its homology to rat SC2(FIGS. 2A and 2B), the transmembrane domain is predicted to includeresidues 255 to 279. SC2H has three potential N-linked glycoslyationsites, all which are on the N-terminal side of the transmembrane domain,at N₁₆₄, N₂₀₈ and N₂₄₇. The localization of the glycosylation sitessuggests that the N-terminal side of the transmembrane domain isextracellular and the C-terminal side of the transmembrane domain facesthe cell interior. Two tyrosines reside on the C-terminal side of thetransmembrane domain at positions Y₂₈₉ and Y₂₉₆ and may serve assubstrates for phosphorylation in signal transduction; Y₂₈₉ is conservedin rat SC2 and Y₂₉₆ is conserved in both the rat and yeast SC2 homologs.The human glycoprotein SC2H has 97% amino acid sequence identity to ratSC2 and 30% sequence identity to yeast SC2 (FIGS. 2A and 2B).

THE SC2H CODING SEQUENCES

The nucleic acid and amino acid sequences of SC2H are shown in FIGS. 1A,1B, and 1C. In accordance with the invention, any nucleic acid sequencewhich encodes the amino acid sequence of SC2H can be used to generaterecombinant molecules which express SC2H. In a specific embodimentdescribed herein, a partial sequence of SC2H was first isolated asIncyte Clone 659029 from a human brain tissue cDNA library (BRAINOT03).

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of SC2H-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.The invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring SC2H, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode SC2H and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring SC2H under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding SC2H or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding SC2H and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

It is now possible to produce a DNA sequence, or portions thereof,encoding an SC2H and its derivatives entirely by synthetic chemistry,after which the synthetic gene may be inserted into any of the manyavailable DNA vectors and cell systems using reagents that are wellknown in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into agene encoding SC2H.

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 1A, 1B, and 1C under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.)incorporated herein by reference, and may be used at a definedstringency.

Altered nucleic acid sequences encoding SC2H which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent SC2H. The protein mayalso show deletions, insertions or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentSC2H. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of SC2H is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine, phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles of SC2H.As used herein, an "allele" or "allelic sequence" is an alternative formof SC2H. Alleles result from a mutation, ie, a change in the nucleicacid sequence, and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one or many allelic forms. Common mutational changes which giverise to alleles are generally ascribed to natural deletions, additionsor substitutions of amino acids. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

Methods for DNA sequencing are well known in the art and employ suchenzymes as the Klenow fragment of DNA polymerase I, SEQUSNASE (USBiochemical Corp, Cleveland Ohio), Taq polymerase (Perkin Elmer, NorwalkConn.), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE amplification system marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the MICROLAB 2200 (Hamilton, Reno Nev.), Peltier thermal cycler(PTC200; MJ Research, Watertown Mass.) and the ABI 377 DNA sequencers(Perkin Elmer).

EXTENDING THE POLYNUCLEOTIDE SEQUENCE

The polynucleotide sequence encoding SC2H may be extended utilizingpartial nucleotide sequence and various methods known in the art todetect upstream sequences such as promoters and regulatory elements.Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose"restriction-site" polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genomic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia T et al (1988) Nucleic AcidsRes 16:8186). The primers may be designed using OLIGO 4.06 primeranalysis software (1992; National Biosciences Inc, Plymouth Minn.), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Capture PCR Lagerstrom M et al (1991) PCR Methods Applic 1:111-19) is amethod for PCR amplification of DNA fragments adjacent to a knownsequence in human and yeast artificial chromosome DNA. Capture PCR alsorequires multiple restriction enzyme digestions and ligations to placean engineered double-stranded sequence into an unknown portion of theDNA molecule before PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker J D et al (1991; Nucleic Acids Res 19:3055-60). Additionally,one can use PCR, nested primers and PromoterFinder libraries to walk ingenomic DNA (PROMOTERFINDER (Clontech, Palo Alto Calif.). This processavoids the need to screen libraries and is useful in finding intron/exonjunctions.

Preferred libraries for screening for full length cDNAs are ones thathave been size-selected to include larger cDNAs. Also, random primedlibraries are preferred in that they will contain more sequences whichcontain the 5' and upstream regions of genes. A randomly primed librarymay be particularly useful if an oligo d(T) library does not yield afull-length cDNA. Genomic libraries are useful for extension into the 5'nontranslated regulatory region.

Capillary electrophoresis may be used to analyze the size or confirm thenucleotide sequence of sequencing or PCR products. Systems for rapidsequencing are available from Perkin Elmer, Beckman Instruments(Fullerton Calif.), and other companies. Capillary sequencing may employflowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity is converted to electrical signal usingappropriate software (eg. GENOTYPER™ and SEQUENCE NAVIGATOR from PerkinElmer) and the entire process from loading of samples to computeranalysis and electronic data display is computer controlled. Capillaryelectrophoresis is particularly suited to the sequencing of small piecesof DNA which might be present in limited amounts in a particular sample.The reproducible sequencing of up to 350 bp of M13 phage DNA in 30 minhas been reported (Ruiz-Martinez M C et al (1993) Anal Chem 65:2851-8).

EXPRESSION OF THE NUCLEOTIDE SEQUENCE

In accordance with the present invention, polynucleotide sequences whichencode SC2H, fragments of the polypeptide, fusion proteins or functionalequivalents thereof may be used in recombinant DNA molecules that directthe expression of SC2H in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequence,may be used to clone and express SC2H. As will be understood by those ofskill in the art, it may be advantageous to produce SC2H-encodingnucleotide sequences possessing non-naturally occurring codons. Codonspreferred by a particular prokaryotic or eukarycotic host (Murray E etal (1989) Nuc Acids Res 17:477-508) can be selected, for example, toincrease the rate of SC2H expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence.

The nucleotide sequences of the present invention can be engineered inorder to alter a coding sequence of SC2H for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, etc.

In another embodiment of the invention, a natural, modified orrecombinant nucleotide sequence encoding SC2H may be ligated to aheterologous sequence to encode a fusion protein. For example, forscreening of peptide libraries for inhibitors of SC2H activity, it maybe useful to encode a chimeric SC2H protein that is recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between an SC2H sequence and theheterologous protein sequence, so that the SC2H may be cleaved andsubstantially purified away from the heterologous moiety.

In an alternate embodiment of the invention, the coding sequence forSC2H may be synthesized, whole or in part, using chemical methods wellknown in the art (see Caruthers M H et al (1980) Nucleic Acids Symp Ser(7): 215-23, Horn T et al (1980) Nucleic Acids Symp Ser (7): 225-232,etc). Alternatively, the protein itself could be produced using chemicalmethods to synthesize an SC2H amino acid sequence, whole or in part. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge J Y et al (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A peptidesynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (eg, Creighton (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co, NewYork N.Y.). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (eg, the Edman degradationprocedure; Creighton, supra). Additionally the amino acid sequence ofSC2H, or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

EXPRESSION SYSTEMS

In order to express a biologically active SC2H, the nucleotide sequenceencoding SC2H or its functional equivalent, is inserted into anappropriate expression vector, ie, a vector which contains the necessaryelements for the transcription and translation of the inserted codingsequence.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing an SC2H coding sequence andappropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y. and Ausubel F M et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y.

A variety of expression vector/host systems may be utilized to containand express an SC2H coding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (eg, baculovirus); plant cell systemstransfected with virus expression vectors (eg, cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

The "control elements" or "regulatory sequences" of these systems varyin their strength and specificities and are those nontranslated regionsof the vector, enhancers, promoters, and 3' untranslated regions, whichinteract with host cellular proteins to carry out transcription andtranslation. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla Calif.) orPSPORT1 (Life Technologies, Rockville Md.) and ptrp-lac hybrids and thelike may be used. The baculovirus polyhedrin promoter may be used ininsect cells. Promoters or enhancers derived from the genomes of plantcells (eg, heat shock, RuBisCo; and storage protein genes) or from plantviruses (eg, viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from the mammalian genes orfrom mammalian viruses are most appropriate. If it is necessary togenerate a cell line that contains multiple copies of SC2H, vectorsbased on SV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for SC2H. For example, when largequantities of SC2H are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asPBLUESCRIPT (Stratagene), in which the SC2H coding sequence may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al (supra) andGrant et al (1987) Methods in Enzymology 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding SC2H may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMV(Brisson et al (1984) Nature 310:511-514) may be used alone or incombination with the omega leader sequence from TMV (Takamatsu et al(1987) EMBO J 6:307-311). Alternatively, plant promoters such as thesmall subunit of RuBisCo (Coruzzi et al (1984) EMBO J 3:1671-1680;Broglie et al (1984) Science 224:838-843); or heat shock promoters(Winter J and Sinibaldi RM (1991) Results Probl Cell Differ 17:85-105)may be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. For reviewsof such techniques, see Hobbs S or Murry L E in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill New York N.Y., pp 191-196 orWeissbach and Weissbach (1988) Methods for Plant Molecular Biology,Academic Press, New York N. Y., pp 421-463.

An alternative expression system which could be used to express SC2H isan insect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The SC2Hcoding sequence may be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the SC2H coding sequence will renderthe polyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses are then used to infect S. frugiperdacells or Trichoplusia larvae in which SC2H is expressed (Smith et al(1983) J Virol 46:584; Engelhard E K et al (1994) Proc Nat Acad Sci91:3224-7).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence for SC2H may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingSC2H in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci81:3655-59). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals nay also be required for efficienttranslation of an SC2H sequence. These signals include the ATGinitiation codon and adjacent sequences. In cases where nucleic acidencoding SC2H, its initiation codon and upstream sequences are insertedinto the appropriate expression vector, no additional translationalcontrol signals may be needed. However, in cases where only codingsequence, or a portion thereof, is inserted, exogenous transcriptionalcontrol signals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf Det al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987)Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressSC2H may be transformed using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt,which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins, βglucuronidase and its substrate, GUS, and luciferase and its substrate,luciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes C A et al (1995)Methods Mol Biol 55:121-131).

IDENTIFICATION OF TRANSFORMANTS CONTAINING THE POLYNUCLEOTIDE SEQUENCE

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the SC2H polynucleotide sequence isinserted within a marker gene sequence, recombinant cells containingSC2H can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with an SC2Hsequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem SC2H as well.

Alternatively, host cells which contain the coding sequence for SC2H andexpress SC2H may be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassaytechniques which include membrane, solution, or chip based technologiesfor the detection and/or quantification of the nucleic acid or protein.

The presence of the polynucleotide sequence encoding SC2H can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of SC2H-encoding nucleotides. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the SC2H sequence to detect transformants containingSC2H DNA or RNA. As used herein "oligonucleotides" or "oligomers" referto a nucleic acid sequence of at least about 10 nucleotides and as manyas about 60 nucleotides, preferably about 15 to 30 nucleotides, and morepreferably about 20-25 nucleotides which can be used as a probe oramplimer.

A variety of protocols for detecting and measuring the expression ofSC2H, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson SC2H is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox DE et al (1983, J Exp Med 158:1211-1226).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to SC2H include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the SC2H sequence, or any portion of it, maybe cloned into a vector for the production of an mRNA probe. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 incorporated herein by reference.

PURIFICATION OF SC2H

Host cells transformed with an SC2H-encoding nucleotide sequence may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be contained intracellularly or secreted dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing SC2H can be designedfor efficient production and proper transmembrane insertion of SC2H intoa prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may join SC2H to nucleotide sequence encoding apolypeptide domain which will facilitate purification of solubleproteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53; of discussionof vectors infra containing fusion proteins).

SC2H may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and SC2H is useful to facilitate purification. One suchexpression vector provides for expression of a fusion proteincompromising an SC2H and contains nucleic acid encoding 6 histidineresidues followed by thioredoxin and an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography as described in Porath et al (1992) ProteinExpression and Purification 3: 263-281) while the enterokinase cleavagesite provides a means for purifying the glycoprotein from the fusionprotein.

In addition on to recombinant production, fragments of SC2H may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart en al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, SanFrancisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, using anABI 431A peptide synthesizer (Perkin Elmer, Foster City Calif.) inaccordance with the instructions provided by the manufacturer. Variousfragments of SC2H may be chemically synthesized separately and combinedusing chemical methods to produce the full length molecule.

USES OF SC2H

The rationale for the use of polynucleotide and polypeptide sequencesdisclosed herein is based in part on the structural homology among thenovel SC2H and rat and yeast SC2 glycoproteins.

Synapse formation or maintenance facilitated by SC2H may influencesynaptic density and affect the release of neurotransmitters or thepropagation of nerve impulses. Therefore, a diagnostic test for alteredexpression of SC2H can accelerate diagnosis and proper treatment ofconditions caused by altered or decreased synaptic contacts or impairedneurotransmitter release, such as Alzheimer's disease, neuromuscularconditions such as Parkinson's disease, Huntington's disease, ALS, orother physiological or pathological problems associated with abnormalneuronal function.

Viral pathogens attach to host cell surfaces by the interaction of viralenvelope glycoproteins with host membrane glycoproteins. This viralattachment is the first step in the infective cycle for numerouspathogens, including those which attack the nervous system such aspoliovirus, rabies, herpes simplex, and HIV. Invasion of viral pathogensmay be controlled by preventing the interaction of viral envelopeglycoproteins with host-cell SC2H glycoprotein.

SC2H or its soluble extracellular domain can be used to identifyspecific cell surface or viral envelope glycoproteins with which SC2Hinteracts. The extracellular domain of SC2H may be used as an inhibitorof virus-host cell attachment by binding to the viral envelopeglycoproteins and thus blocking virus attachment to host membrane SC2H.Alternatively, antibodies raised against the extracellular domain ofSC2H may inhibit interaction of the viral protein with SC2H, therebyblocking virus-host attachment.

In some instances, for instance overexpression associated with disorderssuch as Alzheimer's disease, it may be advantageous to suppress SC2Hexpression. SC2H expression could be suppressed by administration ofSC2H antisense oligonucleotides. Alternatively, specific antibodiesagainst SC2H may be introduced to treat diseases or conditionsassociated with abnormal SC2H expression.

SC2H ANTIBODIES

SC2H-specific antibodies are useful for the diagnosis of conditions anddiseases associated with expression of SC2H. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments and fragments produced by a Fab expressionlibrary. Neutralizing antibodies, ie, those which inhibit dimerformation, are especially preferred for diagnostics and therapeutics.

SC2H for antibody induction does not require biological activity;however, the protein fragment, or oligopeptide must be antigenic.Peptides used to induce specific antibodies may have an amino acidsequence consisting of at least five amino acids, preferably at least 10amino acids. Preferably, they should mimic a portion of the amino acidsequence of the natural protein and may contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofSC2H amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and antibody produced against the chimericmolecule. Procedures well known in the art can be used for theproduction of antibodies to SC2H.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with SC2H or anyportion, fragment or oligopeptide which retains immunogenic properties.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium paryum are potentiallyuseful human adjuvants.

Monoclonal antibodies to SC2H may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Koehler and Milstein (1975 Nature256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983)Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030)and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodiesand Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce SC2H-specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for SC2H mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al (1989) Science 256:1275-1281).

A variety of protocols for competitive binding or immunoradiometricassays using either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between SC2H and its specificantibody and the measurement of complex formation. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo noninterfering epitopes on a specific SC2H protein is preferred, buta competitive binding assay may also be employed. These assays aredescribed in Maddox D E et al 1983, J Exp Med 158:1211).

DIAGNOSTIC ASSAYS USING SC2H SPECIFIC ANTIBODIES

Particular SC2H antibodies are useful for the diagnosis of conditions ordiseases characterized by expression of SC2H or in assays to monitorpatients being treated with SC2H, agonists or inhibitors. Diagnosticassays for SC2H include methods utilizing the antibody and a label todetect SC2H in human body fluids or extracts of cells or tissues. Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Frequently, the polypeptides and antibodies willbe labeled by joining them, either covalently or noncovalently, with areporter molecule. A wide variety of reporter molecules are known,several of which were described above.

A variety of protocols for measuring SC2H, using either polyclonal ormonoclonal antibodies specific for the respective protein are known inthe art. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on SC2H is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D E et al (1983, J Exp Med 158:1211).

In order to provide a basis for diagnosis, normal or standard values forSC2H expression must be established. This is accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with antibody to SC2H under conditions suitable for complexformation which are well known in the art. The amount of standardcomplex formation may be quantified by comparing various artificialmembranes containing known quantities of SC2H with both control anddisease samples from biopsied tissues. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by disease. Deviation betweenstandard and subject values establishes the presence of disease state.

DRUG SCREENING

SC2H, its catalytic or immunogenic fragments or oligopeptides thereof,can be used for screening therapeutic compounds in any of a variety ofdrug screening techniques. The fragment employed in such a test may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenSC2H and the agent being tested, may be measured.

Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe SC2H is described in detail in "Determination of Amino Acid SequenceAntigenicity" by Geysen HN, WO Application 84/03564, published on Sep.13, 1984, and incorporated herein by reference. In summary, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with fragments of SC2H and washed. Bound SC2His then detected by methods well known in the art. Substantiallypurified SC2H can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding SC2Hspecifically compete with a test compound for binding SC2H. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with SC2H.

USES OF THE POLYNUCLEOTIDE ENCODING SC2H

A polynucleotide encoding SC2H, or any part thereof, may be used fordiagnostic and/or therapeutic purposes. For diagnostic purposes, theSC2H of this invention may be used to detect and quantitate geneexpression in biopsied tissues in which expression of SC2H may beimplicated. The diagnostic assay is useful to distinguish betweenabsence, presence, and excess expression of SC2H and to monitorregulation of SC2H levels during therapeutic intervention. Included inthe scope of the invention are oligonucleotide sequences, antisense RNAand DNA molecules, and PNAs.

Another aspect of the subject invention is to provide for hybridizationor PCR probes which are capable of detecting polynucleotide sequences,including genomic sequences, encoding SC2H or closely related molecules.The specificity of the probe, whether it is made from a highly specificregion, eg, 10 unique nucleotides in the 5' regulatory region, or a lessspecific region, eg, especially in the 3' region, and the stringency ofthe hybridization or amplification maximal, high, intermediate or low)will determine whether the probe identifies only naturally occurringSC2H, alleles or related sequences.

Probes may also be used for the detection of related sequences andshould preferably contain at least 50% of the nucleotides from any orthese SC2H encoding sequences. The hybridization probes of the subjectinvention may be derived from the nucleotide sequence of SEQ ID NO:2 orfrom genomic sequence including promoter, enhancer elements and intronsof the naturally occurring SC2H. Hybridization probes may be labeled bya variety of reporter groups, including radionuclides such as ³² P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled to the probevia avidin/biotin coupling systems, and the like.

Other means for producing specific hybridization probes for SC2H DNAsinclude the cloning of nucleic acid sequences encoding SC2H or SC2Hderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art and are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate radioactively labeled nucleotides.

Diagnostics

Polynucleotide sequences encoding SC2H may be used for the diagnosis ofconditions or diseases with which the expression of SC2H is associated.For example, polynucleotide sequences encoding SC2H may be used inhybridization or PCR assays of fluids or tissues from biopsies to detectSC2H expression. The form of such qualitative or quantitative methodsmay include Southern or northern analysis, dot blot or othermembrane-based technologies; PCR technologies; dip stick, pin, chip andELISA technologies. All of these techniques are well known in the artand are the basis of many commercially available diagnostic kits.

The SC2H nucleotide sequence disclosed herein provide the basis forassays that detect activation or induction associated with inflammationor disease. The SC2H nucleotide sequence may be labeled by methods knownin the art and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridization complexes. Afteran incubation period, the sample is washed with a compatible fluid whichoptionally contains a dye (or other label requiring a developer) if thenucleotide has been labeled with an enzyme. After the compatible fluidis rinsed off, the dye is quantitated and compared with a standard. Ifthe amount of dye in the biopsied or extracted sample is significantlyelevated over that of a comparable control sample, the nucleotidesequence has hybridized with nucleotide sequences in the sample, and thepresence of elevated levels of SC2H nucleotide sequences in the sampleindicates the presence of the associated inflammation and/or disease.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regime in animal studies, in clinical trials, orin monitoring the treatment of an individual patient. In order toprovide a basis for the diagnosis of disease, a normal or standardprofile for SC2H expression must be established. This is accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with SC2H, or a portion thereof, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of SC2H run in the sameexperiment where a known amount of substantially purified SC2H is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from patients afflicted with SC2H-associateddiseases. Deviation between standard and subject values is used toestablish the presence of disease.

Once disease is established, a therapeutic agent is administered and atreatment profile is generated. Such assays may be repeated on a regularbasis to evaluate whether the values in the profile progress toward orreturn to the normal or standard pattern. Successive treatment profilesmay be used to show the efficacy of treatment over a period of severaldays or several months.

Polymerase Chain Reaction (PCR) as described in U.S. Pat. Nos. 4,683,195and 4,965,188 provides additional uses for oligonucleotides based uponthe SC2H sequence. Such oligomers are generally chemically synthesized,but they may be generated enzymatically or produced from a recombinantsource. Oligomers generally comprise two nucleotide sequences, one withsense orientation (5'→3') and one with antisense (3'←5'), employed underoptimized conditions for identification of a specific gene or condition.The same two oligomers, nested sets of oligomers, or even a degeneratepool of oligomers may be employed under less stringent conditions fordetection and/or quantitation of closely related DNA or RNA sequences.

Additionally, methods which may be used to quantitate the expression ofa particular molecule include radiolabeling (Melby P C et al 1993 JImmunol Methods 159:235-44) or biotinylating (Duplaa C et a 1993 AnalBiochem 229-36) nucleotides, coamplification of a control nucleic acid,and standard curves onto which the experimental results areinterpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

Therapeutic Use

Based upon its homology to the gene encoding rat SC2 and its expressionprofile, the SC2H polynucleotide disclosed herein may provide the basisfor the design of molecules for the treatment of diseases such asAlzheimer's disease, Huntington disease, Parkinson disease and ALS andfor the prevention of invasion by viral pathogens such as HIV, rabies,and poliovirus.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisenseSC2H. See, for example, the techniques described in Sambrook et al(supra) and Ausubel et al (supra).

The polynucleotides comprising full length cDNA sequence and/or itsregulatory elements enable researchers to use SC2H as an investigativetool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem60:631-652) regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers, or larger fragments,can be designed from various locations along the coding or controlregions.

Genes encoding SC2H can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a desired SC2Hfragment. Such constructs can flood cells with untranslatable sense orantisense sequences. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until all copiesare disabled by endogenous nucleases. Transient expression may last fora month or more with a non-replicating vector (Mettler I, personalcommunication) and even longer if appropriate replication elements arepart of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA or PNA, to the control regionsof SC2H, ie, the promoters, enhancers, and introns. Oligonucleotidesderived from the transcription initiation site, eg, between -10 and +10regions of the leader sequence, are preferred. The antisense moleculesmay also be designed to block translation of mRNA by preventing thetranscript from binding to ribosomes. Similarly, inhibition can beachieved using "triple helix" base-pairing methodology. Triple helixpairing compromises the ability of the double helix to open sufficientlyfor the binding of polymerases, transcription factors, or regulatorymolecules. Recent therapeutic advances using triplex DNA were reviewedby Gee J E et al (In: Huber B E and B I Carr (1994) Molecular andImmunologic Approaches, Futura Publishing Co, Mt Kisco N.Y.).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of RNA encoding SC2H.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding SC2H. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Methods for introducing vectors into cells or tissues include thosemethods discussed infra and which are equally suitable for in vivo, invitro and ex vivo therapy. For ex vivo therapy, vectors are introducedinto stem cells taken from the patient and clonally propagated forautologous transplant back into that same patient as presented in U.S.Pat. Nos. 5,399,493 and 5,437,994, disclosed herein by reference.Delivery by transfection and by liposome are quite well known in theart.

Furthermore, the nucleotide sequences for SC2H disclosed herein may beused in molecular biology techniques that have not yet been developed,provided the new techniques rely on properties of nucleotide sequencesthat are currently known, including but not limited to such propertiesas the triplet genetic code and specific base pair interactions.

DETECTION AND MAPPING OF RELATED POLYNUCLEOTIDE SEQUENCES

The nucleic acid sequence for SC2H can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C M (1993; Blood Rev 7:127-34) and Trask B J (1991;Trends Genet 7:149-54).

The technique of fluorescent in situ hybridization of chromosome spreadshas been described, among other places, in Verma et al (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofan SC2H on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. For example, an STSbased map of the human genome was recently published by theWhitehead-MIT Center for Genomic Research (Hudson T J et al (1995)Science 270:1945-1954). Often the placement of a gene on the chromosomeof another mammalian species such as mouse (Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995) may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22-23 (Gatti et al (988) Nature336:577-580), any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc. amongnormal, carrier or affected individuals.

PHARMACEUTICAL COMPOSITIONS

The present invention relates to pharmaceutical compositions which maycomprise nucleotides, proteins, antibodies, agonists, antagonists, orinhibitors, alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. Any of these molecules canbe administered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the present invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

Administration of Pharmaceutical Compositions

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie, dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Manufacture and Storage

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, eg, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of SC2H, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, eg, of neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors which ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, eg, ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The BRAINOT03 cDNA library was constructed from normal brain tissueremoved from a 26 year old male. The frozen tissue was homogenized andlysed using a POLYTRON homogenizer CPT-3000; (Brinkmann Instruments,Westbury N.J.). The reagents and extraction procedures were used assupplied in the Stratagene RNA Isolation Kit (Cat. #200345; Stratagene).The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotorin a L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000rpm at ambient temperature. The RNA was extracted once with phenolchloroform pH 8.0, once with acid phenol pH 4.0, precipitated using 0.3M sodium acetate and 2.5 volumes of ethanol, resuspended in water andDNase treated for 15 min at 37° C. The RNA was isolated using theOLIGOTEX kit (QIAGEN Inc, Chatsworth Calif.) and used to construct thecDNA library.

The RNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system (Cat. #418248-013; Gibco/BRL). cDNAs werefractionated on a SEPHAROSE CL4B column (Cat. #275105, Pharmacia), andthose cDNAs exceeding 400 bp were ligated into PSPORT 1. The plasmidPSPORT 1 was subsequently transformed into DH5a competent cells (Cat.#18258-012, Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the MINIPREPKit (Cat. #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Cat. #22711, Life Technologies, withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria werecultured for 24 hours after the wells were inoculated and then lysedwith 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R rotor (Beckman Instruments) at 2900 rpm for 5 min wasperformed before the contents of the block were added to the primaryfilter plate; and 4) the optional step of adding isopropanol to TRISbuffer was not routinely performed. After the last step in the protocol,samples were transferred to a 96-well block for storage.

The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975;J Mol Biol 94:441f), using a MICROLAB 2200 (Hamilton, Reno Nev.) incombination with four Peltier thermal cyclers (PTC200; MJ Research,Watertown Mass.) and ABI PRISM 377 Sequencing system (Perkin Elmer), andreading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT 670sequence analysis system (Perkin Elmer). In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 sequence analysis system (Perkin Elmer) in a way similar tothat used in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul S F(1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful In determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al. supra).

Analogous computer techniques use BLAST (Altschul SF 1993 and 1990,supra) to search for identical or related molecules in nucleotidedatabases such as GenBank or the LIFESEQ database (Incyte,Pharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## and it takes into account both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

V Extension of SC2H to Full Length or to Recover Regulatory Elements

The nucleic acid sequence encoding full length SC2H (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5' sequences fromgenomic libraries. One primer is synthesized to initiate extension inthe antisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers allow the extension ofthe known SC2H nucleotide sequence "outward" generating ampliconscontaining new, unknown nucleotide sequence for the region of interest(U.S. patent application Ser. No. 08/487,112, filed Jun. 7, 1995,specifically incorporated by reference). The initial primers aredesigned from the cDNA using OLIGO 4.06 primer analysis software(National Biosciences), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier thermal cycler (PTC200; MJ Research,Watertown Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                   Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       94° C. for 15 sec                                         Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       94° C. for 15 sec                                         Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. for 8 min                                          Step 13      4° C. (and holding)                                       ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products were selected and cut out of the gel.Further purification involves using a commercial gel extraction methodsuch as the QIAQUICK Kit (QIAGEN Inc). After recovery of the DNA, Klenowenzyme was used to trim single-stranded, nucleotide overhangs creatingblunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                           Step 2     94° C. for 20 sec                                           Step 3     55° C. for 30 sec                                           Step 4     72° C. for 90 sec                                           Step 5     Repeat steps 2-4 for an additional 29 cycles                       Step 6     72° C. for 180 sec                                          Step 7     40° C. (and holding)                                        ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides,consisting of about 20 base-pairs, is specifically described,essentially the same procedure is used with larger cDNA fragments.Oligonuclectides are designed using state-of-the-art software such asOLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of eacholigomer and 250 mCi of [γ-³² P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN, Boston Mass.).The labeled oligonucleotides are substantially purified with SEPHADEXG-25 super fine resin column (Pharmacia). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots or the blots arein a PHOSPHOIMAGER cassette (Molecular Dynamics, Sunnyvale Calif.) forseveral hours, hybridization patterns are compared.

VII Antisense Molecules

The nucleotide sequence encoding SC2H, or any part thereof, is used toinhibit in vivo or in vitro expression of naturally occurring SC2H.Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequence of SC2H as shown in FIGS. 1A, 1B, and 1C is used to inhibitexpression of naturally occurring SC2H. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A, 1B, and 1C and used either to inhibit transcription bypreventing promoter binding to the upstream nontranslated sequence ortranslation of an SC2H transcript by preventing the ribosome frombinding. Using an appropriate portion of the leader and 5' sequence ofSEQ ID NO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal orearly coding sequence of the polypeptide as shown in FIGS. 1A, 1B, and1C.

VIII Expression of SC2H

Expression of the extracellular domain of SC2H is accomplished bysubcloning the cDNAs into appropriate vectors and transfecting thevectors into host cells. In this case, the cloning vector, PSPORT 1(Life Technologies), previously used for the generation of the cDNAlibrary is used to express SC2H in E. coli. Upstream of the cloningsite, this vector contains a promoter for β-galactosidase, followed bysequence containing the amino-terminal Met and the subsequent 7 residuesof β-galactosidase. Immediately following these eight residues is abacteriophage promoter useful for transcription and a linker containinga number of unique restriction sites.

Induction of an isolated, transfected bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firstseven residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length SC2H. The signal sequence directs the secretion of theextracellular domain of SC2H into the bacterial growth media which canbe used directly in the following assay for activity.

IX SC2H Activity

The binding activity of SC2H or biologically active fragments thereofmay be assayed by first labeling the SC2H protein or polypeptide with¹²⁵ I Bolton-Hunter reagent (Bolton, A E and Hunter, W M (1973) BiochemJ 133: 529). Candidate ligands (including glycoproteins, virus particlesor whole cells) previously arrayed in the wells of a 96 well plate areincubated with the labeled SC2H, washed and any wells with labeled SC2Hcomplex are assayed by radioactivity quantitation. Data obtained usingdifferent concentrations of SC2H are used to calculate values for thenumber, affinity, and association of SC2H with the candidate ligands.

X Production of SC2H Specific Antibodies

SC2H substantially purified using PAGE electrophoresis (Sambrook, supra)is used to immunize rabbits and to produce antibodies using standardprotocols. The amino acid sequence translated from SC2H is analyzedusing LASERGENE Software (DNAStar Inc) to determine regions of highimmunogenicity and a corresponding oligopolypeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Analysis to select appropriate epitopes, such as those near theC-terminus or in hydrophilic regions (shown in FIG. 3) is described byAusubel F M et al (supra).

Typically, the oligopeptides are 15 residues in length, synthesizedusing an ABI 431A peptide synthesizer using fmoc-chemistry, and coupledto keyhole limpet hemocyanin (KLH, Sigma) by reaction withM-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

XI Purification of Naturally Occurring SC2H Using Specific Antibodies

Naturally occurring or recombinant SC2H is substantially purified byimmunoaffinity chromatography using antibodies specific for SC2H. Animmunoaffinity column is constructed by covalently coupling SC2Hantibody to an activated chromatographic resin such as CnBr-activatedSEPHAROSE (Pharmacia Biotech). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Cellular fractions from cells containing SC2H are prepared bysolubilization of the whole cell and isolation of subcellular fractionsby differential centrifugation, by the addition of detergent, or byother methods well known in the art. Alternatively, soluble SC2Hcontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A fractionated SC2H-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of SC2H (eg, high ionic strengthbuffers in the presence of detergent). The column is eluted underconditions that disrupt antibody/SC2H binding (eg, a buffer of pH 2-3 ora high concentration of a chaotrope such as urea or thiocyanate ion),and SC2H is collected.

XII Identification of Molecules Which Interact with SC2H Labeled SC2H isuseful as a research tool for identification, characterization andpurification of molecules with which it interacts. In one embodiment ofaffinity purification, SC2H is covalently coupled to a chromatographycolumn. Cells and their membranes are extracted, endogenous SC2H isremoved and various SC2H-free subcomponents are passed over the column.SC2H-associated molecules bind to the column by virtue of theirbiological affinity. The SC2H-complex is recovered from the column,dissociated and the recovered molecule is subjected to N-terminalprotein sequencing. This amino acid sequence is then used to identifythe captured molecule or to design degenerate oligonucleotide probes forcloning its gene from an appropriate cDNA library.

In another alternate method, monoclonal antibodies are raised againstSC2H and screened to identify those which inhibit the binding of labeledSC2H. These monoclonal antibodies are then used in affinity purificationor expression cloning of associated molecules.

Other soluble binding molecules are identified in a similar manner.Labeled SC2H is incubated with extracts or biopsied materials derivedfrom cells or tissues such as rheumatoid synovium, lung or cerebellum.After incubation, SC2H complexes (which are larger than the size ofpurified SC2H molecule) are identified by a sizing technique such assize exclusion chromatography or density gradient centrifugation and arepurified by methods known in the art. The soluble binding protein(s) aresubjected to N-terminal sequencing to obtain information sufficient fordatabase identification, if the soluble protein is known, or forcloning, if the soluble protein is unknown.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 308 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: BRAINOT03                                                        (B) CLONE: 659029                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Lys His Tyr Glu Val Glu Ile Leu Asp Al - #a Lys Thr Arg Glu Lys         #                15                                                           - Leu Cys Phe Leu Asp Lys Val Glu Pro His Al - #a Thr Ile Ala Glu Ile         #            30                                                               - Lys Asn Leu Phe Thr Lys Thr His Pro Gln Tr - #p Tyr Pro Ala Arg Gln         #        45                                                                   - Ser Leu Arg Leu Asp Pro Lys Gly Lys Ser Le - #u Lys Asp Glu Asp Val         #    60                                                                       - Leu Gln Lys Leu Pro Val Gly Thr Thr Ala Th - #r Leu Tyr Phe Arg Asp         #80                                                                           - Leu Gly Ala Gln Ile Ser Trp Val Thr Val Ph - #e Leu Thr Glu Tyr Ala         #                95                                                           - Xaa Pro Leu Phe Ile Tyr Leu Leu Phe Tyr Ph - #e Arg Val Pro Phe Ile         #           110                                                               - Tyr Gly His Lys Tyr Asp Phe Thr Ser Ser Ar - #g His Thr Val Val His         #       125                                                                   - Leu Ala Cys Ile Cys His Ser Phe His Tyr Il - #e Lys Arg Leu Leu Glu         #   140                                                                       - Thr Leu Phe Val His Arg Phe Ser His Gly Th - #r Met Pro Leu Arg Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Phe Lys Asn Cys Thr Tyr Tyr Trp Gly Ph - #e Ala Ala Trp Met Ala         #               175                                                           - Tyr Tyr Ile Asn His Pro Leu Tyr Thr Pro Pr - #o Thr Tyr Gly Ala Gln         #           190                                                               - Gln Val Lys Leu Ala Leu Ala Ile Phe Val Il - #e Cys Gln Leu Gly Asn         #       205                                                                   - Phe Ser Ile His Met Ala Leu Arg Asp Leu Ar - #g Pro Ala Gly Ser Lys         #   220                                                                       - Thr Arg Lys Ile Pro Tyr Pro Thr Lys Asn Pr - #o Phe Thr Trp Leu Xaa         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Leu Val Ser Cys Pro Asn Tyr Thr Tyr Gl - #u Val Gly Ser Trp Ile         #               255                                                           - Gly Phe Ala Ile Met Thr Gln Cys Leu Pro Va - #l Ala Leu Phe Ser Leu         #           270                                                               - Val Gly Phe Thr Gln Met Thr Ile Trp Ala Ly - #s Gly Lys His Arg Ser         #       285                                                                   - Tyr Leu Lys Glu Phe Arg Asp Tyr Pro Pro Le - #u Arg Met Pro Ile Ile         #   300                                                                       - Pro Phe Leu Leu                                                             305                                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1137 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: BRAINOT03                                                        (B) CLONE: 659029                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - ATCGCTGCGG TTGCGAGCGC TGTAGGGAGC CTGTGCTGTG CCGCGCAGTT AG - #GCAGCAGC         60                                                                          - AGCCGCGGAG CAGTAGCCGC CGTGGGAGGG AGCCATGAAG CATTACGAGG TG - #GAGATTCT        120                                                                          - GGACGCAAAG ACAAGGGAGA AGCTGTGTTT CTTGGACAAG GTGGAGCCCC AC - #GCCACCAT        180                                                                          - TGCGGAGATC AAGAACCTCT TCACTAAGAC CCATCCGCAG TGGTACCCCG CC - #CGCCAGTC        240                                                                          - CCTCCGCCTG GACCCCAAGG GCAAGTCCCT GAAGGATGAG GATGTTCTGC AG - #AAGCTGCC        300                                                                          - CGTGGGCACC ACGGCCACAC TGTACTTCCG GGACCTGGGG GCCCAGATCA GC - #TGGGTGAC        360                                                                          - GGTCTTCCTA ACAGAGTACG CGNGGCCCCT TTTCATCTAC CTGCTCTTCT AC - #TTCCGAGT        420                                                                          - GCCCTTCATC TATGGCCACA AATATGACTT TACGTCCAGT CGGCATACAG TG - #GTGCACCT        480                                                                          - CGCCTGCATC TGTCACTCAT TCCACTACAT CAAGCGCCTG CTGGAGACGC TC - #TTCGTGCA        540                                                                          - CCGCTTCTCC CATGGCACTA TGCCTTTGCG CAACATCTTC AAGAACTGCA CC - #TACTACTG        600                                                                          - GGGCTTCGCC GCGTGGATGG CCTATTACAT CAATCACCCT CTCTACACTC CC - #CCTACCTA        660                                                                          - CGGAGCTCAG CAGGTGAAAC TGGCGCTCGC CATCTTTGTG ATCTGCCAGC TC - #GGCAACTT        720                                                                          - CTCCATCCAC ATGGCCCTGC GGGACCTGCG GCCCGCTGGG TCCAAGACGC GG - #AAGATCCC        780                                                                          - ATACCCCACC AAGAACCCCT TCACGTGGCT CNTTCTGCTG GTGTCCTGCC CC - #AACTACAC        840                                                                          - CTACGAGGTG GGGTCCTGGA TCGGTTTCGC CATCATGACG CAGTGTCTCC CA - #GTGGCCCT        900                                                                          - GTTCTCCCTG GTGGGCTTCA CCCAGATGAC CATCTGGGCC AAGGGCAAGC AC - #CGCAGCTA        960                                                                          - CCTGAAGGAG TTCCGGGACT ACCCGCCCCT GCGCATGCCC ATCATCCCCT TC - #CTGCTCTG       1020                                                                          - AGCGCTCACC CCTGCTGAGG CTCAGCCCCT CAACCCGGTG GCATTCTGGG GG - #AGGAGTGG       1080                                                                          - GGCCCACAGC TCTCCAGCAC CCGGAATAAA GCCCGCCTGC CCCAGTCGGA AA - #AAAAA          1137                                                                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 308 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 256993                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Lys His Tyr Glu Val Glu Ile Arg Asp Al - #a Lys Thr Arg Glu Lys         #                15                                                           - Leu Cys Phe Leu Asp Lys Val Glu Pro Gln Al - #a Thr Ile Ser Glu Ile         #            30                                                               - Lys Thr Leu Phe Thr Lys Thr His Pro Gln Tr - #p Tyr Pro Ala Arg Gln         #        45                                                                   - Ser Leu Arg Leu Asp Pro Lys Gly Lys Ser Le - #u Lys Asp Glu Asp Val         #    60                                                                       - Leu Gln Lys Leu Pro Val Gly Thr Thr Ala Th - #r Leu Tyr Phe Arg Asp         #80                                                                           - Leu Gly Ala Gln Ile Ser Trp Val Thr Val Ph - #e Leu Thr Glu Tyr Ala         #                95                                                           - Gly Pro Leu Phe Ile Tyr Leu Leu Phe Tyr Ph - #e Arg Val Pro Phe Ile         #           110                                                               - Tyr Gly Arg Lys Tyr Asp Phe Thr Ser Ser Ar - #g His Thr Val Val His         #       125                                                                   - Leu Ala Cys Met Cys His Ser Phe His Tyr Il - #e Lys Arg Leu Leu Glu         #   140                                                                       - Thr Leu Phe Val His Arg Phe Ser His Gly Th - #r Met Pro Leu Arg Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Phe Lys Asn Cys Thr Tyr Tyr Trp Gly Ph - #e Ala Ala Trp Met Ala         #               175                                                           - Tyr Tyr Ile Asn His Pro Leu Tyr Thr Pro Pr - #o Thr Tyr Gly Val Gln         #           190                                                               - Gln Val Lys Leu Ala Leu Ala Ile Phe Val Il - #e Cys Gln Leu Gly Asn         #       205                                                                   - Phe Ser Ile His Met Ala Leu Arg Asp Leu Ar - #g Pro Ala Gly Ser Lys         #   220                                                                       - Thr Arg Lys Ile Pro Tyr Pro Thr Lys Asn Pr - #o Phe Thr Trp Leu Phe         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Leu Val Ser Cys Pro Asn Tyr Thr Tyr Gl - #u Val Gly Ser Trp Ile         #               255                                                           - Gly Phe Ala Ile Met Thr Gln Cys Val Pro Va - #l Ala Leu Phe Ser Leu         #           270                                                               - Val Gly Phe Thr Gln Met Thr Ile Trp Ala Ly - #s Gly Lys His Arg Ser         #       285                                                                   - Tyr Leu Lys Glu Phe Arg Asp Tyr Pro Pro Le - #u Arg Met Pro Ile Ile         #   300                                                                       - Pro Phe Leu Leu                                                             305                                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 310 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 1077162                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Pro Ile Thr Ile Lys Ser Arg Ser Lys Gl - #y Leu Arg Asp Thr Glu         #                15                                                           - Ile Asp Leu Ser Lys Lys Pro Thr Leu Asp As - #p Val Leu Lys Lys Ile         #            30                                                               - Ser Ala Asn Asn His Asn Ile Ser Lys Tyr Ar - #g Ile Arg Leu Thr Tyr         #        45                                                                   - Lys Lys Glu Ser Lys Gln Val Pro Val Ile Se - #r Glu Ser Phe Phe Gln         #    60                                                                       - Glu Glu Ala Asp Asp Ser Met Glu Phe Phe Il - #e Lys Asp Leu Gly Pro         #80                                                                           - Gln Ile Ser Trp Arg Leu Val Phe Phe Cys Gl - #u Tyr Leu Gly Pro Val         #                95                                                           - Leu Val His Ser Leu Phe Tyr Tyr Leu Ser Th - #r Ile Pro Thr Val Val         #           110                                                               - Asp Arg Trp His Ser Ala Ser Ser Asp Tyr As - #n Pro Phe Leu Asn Arg         #       125                                                                   - Val Ala Tyr Phe Leu Ile Leu Gly His Tyr Gl - #y Lys Arg Leu Phe Glu         #   140                                                                       - Thr Leu Phe Val His Gln Phe Ser Leu Ala Th - #r Met Pro Ile Phe Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Phe Lys Asn Cys Phe His Tyr Trp Val Le - #u Ser Gly Leu Ile Ser         #               175                                                           - Phe Gly Tyr Phe Gly Tyr Gly Phe Pro Phe Gl - #y Asn Ala Lys Leu Phe         #           190                                                               - Lys Tyr Tyr Ser Tyr Leu Lys Leu Asp Asp Le - #u Ser Thr Leu Ile Gly         #       205                                                                   - Leu Phe Val Leu Ser Glu Leu Trp Asn Phe Ty - #r Cys His Ile Lys Leu         #   220                                                                       - Arg Leu Trp Gly Asp Tyr Gln Lys Lys His Gl - #y Asn Ala Lys Ile Arg         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Pro Leu Asn Gln Gly Ile Phe Asn Leu Ph - #e Val Ala Pro Asn Tyr         #               255                                                           - Thr Phe Glu Val Trp Ser Trp Ile Trp Phe Th - #r Phe Val Phe Lys Phe         #           270                                                               - Asn Leu Phe Ala Val Leu Phe Leu Thr Val Se - #r Thr Ala Gln Met Tyr         #       285                                                                   - Ala Trp Ala Gln Lys Lys Asn Lys Lys Tyr Hi - #s Thr Arg Arg Ala Phe         #   300                                                                       - Leu Ile Pro Phe Val Phe                                                     305                 3 - #10                                                   __________________________________________________________________________

What is claimed is:
 1. A purified polypeptide comprising the amino acidsequence of SEQ ID NO:1.
 2. The purified polypeptide of claim 1 havingstructural characteristics of the class of glycoproteins including SC2.3. A composition comprising a purified glycoprotein comprising SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.